Project Summary My laboratory studies genes and cell biological mechanisms that form, shape and protect epithelial tubes, with a focus on very narrow unicellular tubes and apical extracellular matrix (aECM). Both are topics relevant to human diseases involving capillaries and other narrow tubes, but are challenging to study in most animal models due to difficulties in imaging the relevant structures and molecules. The nematode C. elegans allows us to circumvent these challenges by permitting imaging of live animals at sub-cellular resolution, combined with powerful genetic approaches to identify relevant genes and pathways. Our recent work showed that a unicellular tube is shaped by: (i) intracellular membrane trafficking events that rely on a fusogen protein (AFF-1) better known for its roles in cell-cell fusion; and (ii) a newly discovered type of aECM that precedes the cuticle and contains proteins similar to those found in or near some mammalian ECMs (e.g. those with leucine-rich repeat, plasminogen, zona pellucida, mucin and/or nidogen domains). This aECM also relies on putative lipid transporters of the lipocalin, scavenger receptor B (SCARB) and Patched-related families. Our research program in the next five years will follow up on these advances to better understand the specific trafficking events involved in building unicellular tubes and the structure and function of the aECM. For example, we will further test the model that AFF-1 mediates endocytic scission to allow membrane to be shuttled from basal to apical surfaces to extend the tube lumen, and we will identify AFF-1 domains and partners involved in this scission function. We will investigate how specific glycoproteins traffic to and assemble within the aECM, how proper aECM lipid content is regulated by transporter families, and how individual aECM components affect the shape and integrity of differently-sized tubes in the organism.